Compositions for the treatment of gluten intolerance and uses thereof

ABSTRACT

The present invention provides compositions for use in the prophylaxis or treatment of a condition arising from gluten intolerance, the compositions including at least partially purified caricain (or a biologically active fragment, analogue or variant thereof) alone or in combination with other suitable enzymes including bromelain, and/or an intestinal extract, as herein described. The present invention also provides methods of using such compositions for the prophylaxis or treatment of a condition arising from gluten intolerance.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/144,549, filed on Jul. 14, 2011, which claims priority toInternational Application No. PCT/AU2010/000006, filed on Jan. 6, 2010,which claims priority to Australian Patent Application No. 2009900164,filed on Jan. 15, 2009. The contents of each application are herebyincorporated by reference in their entirety.

The present invention relates generally to compositions for theprophylaxis or therapy of conditions related to gluten intolerance, anduses thereof.

BACKGROUND

Gluten intolerance is found predominantly in areas where wheat is amajor food source, (e.g., Europe, North America and Australia). In theseareas, the incidence of the disease is about 1 per 100 head ofpopulation (DA Van Heel et al, Gut 2006; 55:1037-1046). The symptoms ofthis condition include abdominal pain, bloating and diarrhoea. In severeand long term cases, such as in coeliac disease, there are inflammatorychanges to the intestinal mucosa, resulting in malabsorption ofnutrients, fatigue, chronic diarrhoea, weight loss, abdominaldistension, anaemia, increased tendency to haemorrhage, as well asincreased risk of gastrointestinal malignancies, such as lymphoma andcarcinoma.

The pathogenesis of gluten intolerance (or coeliac disease, coeliacsprue) appears to have both genetic and environmental factors. Whilstgenetic predisposition is a major factor (about 10% of first degreerelatives are affected), the fact that monozygotic twins have aconcordance rate of only about 75% suggests that environment also playsa part in the development of the disease.

Patients with gluten intolerance characteristically have T cells presentin the intestinal mucosa which recognize certain sequences present intoxic gluten peptides. Evidence suggests that these T cells play acrucial role in the immunopathogenesis of the disease by recognisingpeptides containing specific sequences of amino acids associated withtoxicity. For instance, the proliferation of gliadin-specificHLA-DQ2-restricted T cell clones from the intestine of coeliac diseasepatients can be initiated in vitro by the addition of a water-soluble,partially digested form of gliadin to HLA-DQ2 carryingantigen-presenting cells.

A related disease associated with severe gluten intolerance isdermatitis herpetiformis, which presents as a chronic eruptioncharacterized by clusters of intensely pruritic vesicles, papules, andurticaria-like lesions. Studies have shown that IgA deposits occur inalmost all normal-appearing and perilesional skin. Asymptomaticgluten-sensitive enteropathy is found in 75 to 90% of patients and insome of their relatives. The onset of dermatitis herpetiformis isusually gradual, progressing to severe itching and burning of theaffected surface. Moreover, scratching often obscures the primarylesions with eczematization of nearby skin, leading to an erroneousdiagnosis of eczema.

Gluten is a protein fraction found, for example, in cereal dough, whichcan be subdivided into glutenins and prolamins. Prolamins may also besubclassified as gliadins, secalins, hordeins, and avenins from wheat,rye, barley and oat, respectively. Among gluten proteins with potentialharmful effect to gluten intolerant patients are the storage proteins ofwheat, species of which include Triticum aestivum; Triticum aethiopicum;Triticum baeoticum; Triticum militinae; Triticum monococcum; Triticumsinskajae; Triticum timopheevii; Triticum turgidum; Triticum urartu,Triticum vavilovii; Triticum zhukovskyi; etc. (see, for example, Colot,Genet Eng (NY) 12:225-41, 1990).

Gliadin is the 70% alcohol-soluble protein fraction of wheat gluten.Derived from wheat flour, gliadins can be classified into several groupsaccording to their electrophoretic mobility, including α-type, β-type,γ-type and ω-type. Gliadins are typically rich in glutamine and proline,particularly in the N-terminal part. For example, the first 100 aminoacids of a and γ-gliadins contain about 35% and about 20% of glutamineand proline residues, respectively. Different gliadins are present ineach subcultivar of wheat, with variations in the amino acid sequenceswithin each type. Gliadins are typically characterized by a molecularmass of around 30-50 kDalton and their insolubility in neutral aqueoussolutions. Examples of gliadin sequences include but are not limited towheat α-gliadin sequences, for example as provided in Genbank, accessionnumbers AJ133612; AJ133611; AJ133610; AJ133609; AJ133608; AJ133607;AJ133606; AJ133605; AJ133604; AJ133603; AJ133602; D84341.1; U51307;U51306; U51304; U51303; U50984; and U08287. A sequence of wheat omegagliadin is set forth in Genbank accession number AF280605.

It has been discovered that in gluten intolerant individuals, enzymesnormally present in the small bowel that are necessary for the digestionof gluten are missing. Peptide fragments produced by incompletedigestion of grain protein are toxic to such individuals; the most toxicpeptides being those derived from α-gliadin, or a similar protein calledA-gliadin.

Serine-containing peptides, (containing PSQQ and possibly also QQQPmotifs, as found in residues 11-19 of A-gliadin), appear to have acytotoxic effect. Tyrosine-containing peptides, (containing QQPY and/orQPYP motifs, as found in residues 75-86 of A-gliadin), are associatedwith immunological activity through T-cell mediation and hence,toxicity.

Experiments indicate that active serine-containing peptides like 11-19and active tyrosine-containing peptides like 75-86 are incompletelydigested by mucosal enzymes in patients suffering from coeliac disease.The residual peptide sequences, such as 11-18 and 77-84, are stilltoxic, which suggests that the aetiology of coeliac disease is connectedto defective mucosal digestion and that the pathogenesis of the diseaseresults from the action of the undigested peptides on the mucosa. Thismay ultimately be due to deficiency in a single enzyme in coeliacdisease patients, but at least two different types of peptide residuesbuild up and cause damage to mucosal tissue.

At present, there is no effective therapy for treating the effects ofgluten intolerance other than to impose a gluten-free diet on thepatient. However, due to the number of food products containing eithercereals comprising gluten, or gluten per se, this approach constitutes asevere restriction to the food choices available to a patient. Moreover,although gluten withdrawal has improved the prognosis of glutenintolerant patients, some people still die of the disease, presumablyfrom lymphoreticular disease (especially intestinal lymphoma),particularly in those people who present with severe gluten intoleranceat the outset. It appears that gluten withdrawal diminishes the risk ofdeveloping lymphoreticular disease, whilst apparent clinical remissionis often associated with histologic relapse that is detected only byreview biopsies or by an increased IgA class anti-endomysial antibody(EMA) titre.

In view of the serious and widespread nature of gluten intolerance,improved methods of treating, preventing or ameliorating the effects ofthis condition are needed. Accordingly, it is an aspect of the presentinvention to overcome, or at least partly alleviate, some of theaforementioned problems of the art by providing improved compositionsfor preventing or treating conditions arising from gluten intoleranceand methods for their use.

The discussion of documents, acts, materials, devices, articles and thelike is included in this specification solely for the purpose ofproviding a context for the present invention. It is not suggested orrepresented that any or all of these matters formed part of the priorart base or were common general knowledge in the field relevant to thepresent invention as it existed before the priority date of each claimof this application.

SUMMARY OF THE INVENTION

In one aspect of the present invention, there is provided a compositionfor the prophylaxis or treatment of a condition related to glutenintolerance, the composition including at least partially purifiedcaricain, or a biologically active fragment, analogue or variantthereof.

In one embodiment, caricain is derived from Carica papaya.

In another embodiment, the caricain includes an amino acid sequence asshown in FIG. 1, or a biologically active fragment, analogue or variantthereof.

The composition according to the present invention may further includean animal intestinal enzyme extract, as herein described.

In another embodiment, the composition according to the presentinvention includes bromelain or a biologically active fragment, analogueor variant thereof.

The composition according to the present invention may be formulated asan enterically coated tablet or capsule.

In some embodiments of the present invention, the composition includesat least 15 mg of caricain, or a biologically active fragment, variantand analogue thereof.

In another aspect of the present invention, there is provided acomposition for use in the prophylaxis or treatment of a conditionrelated to gluten intolerance, the composition including a nucleic acidmolecule that encodes recombinant caricain, or a biologically activefragment, analogue or variant thereof. In one embodiment, the nucleicacid molecule includes a nucleotide sequence as shown in FIG. 2, or afunctional equivalent thereof. In another embodiment, the nucleic acidmolecule encodes an amino acid sequence as shown in FIG. 1, or abiologically active fragment, analogue or variant thereof.

The composition according to the present invention may further include anucleic acid molecule that is capable of expressing recombinantbromelain, or a biologically active fragment, analogue or variantthereof.

In another embodiment of the present invention, there is provided acomposition for use in the prophylaxis or treatment of a conditionrelated to gluten intolerance, wherein the composition includes a hostcell that is capable of expressing recombinant caricain, or abiologically active fragment, analogue or variant thereof.

In yet another embodiment of the present invention, the compositionincludes a host cell that is capable of expressing recombinant bromelainor a biologically active fragment, analogue or variant thereof.

In another aspect of the present invention, there is provided a methodfor the prophylaxis or treatment of a condition related to glutenintolerance, the method including administering to a subject in needthereof a composition including at least partially purified caricain, ora biologically active fragment, analogue or variant thereof, as hereindescribed. In some embodiments of the present invention, the compositionincludes at least 15 mg of caricain, or a biologically active fragment,variant and analogue thereof. In some embodiments of the presentinvention, the composition is administered to the subject in needthereof before a meal.

In one embodiment, the method according to the present inventionincludes administering to the subject in need thereof a compositionincluding an intestinal extract.

In yet another embodiment, the method according to the present inventionincludes administering to the subject in need thereof bromelain or abiologically active fragment, analogue or variant thereof.

In another aspect of the present invention, there is provided a methodfor the prophylaxis or treatment of a condition arising from glutenintolerance, the method including administering to a subject in needthereof a nucleic acid molecule, wherein the nucleic acid molecule iscapable of expressing in the subject recombinant caricain, or abiologically active fragment, analogue or variant thereof, as hereindescribed.

In one embodiment, the method according to the present inventionincludes administering to the subject in need thereof a nucleic acidmolecule that is capable of expressing in the subject recombinantbromelain, or a biologically active fragment, analogue or variantthereof.

It is yet another aspect of the present invention to provide a method ofpreparing an article of food derived from a gluten-containing material,the method including treating the article of food or thegluten-containing material with caricain, or a biologically activefragment, analogue or variant thereof, so as to reduce the amount oftoxic gluten-derived oligopeptides present in the article of food. Inone embodiment, the method according to the present invention mayfurther include treating the article of food or the gluten-containingmaterial with bromelain, or a biologically active fragment, analogue orvariant thereof.

FIGURES

FIG. 1 illustrates the primary amino acid sequence of Carica papayacaricain (GenBank Accession No. X66060).

FIG. 2 illustrates the primary amino acid sequence of Carica papayacaricain (GenBank Accession No. X69877).

FIG. 3 illustrates the mRNA sequence of Carica papaya caricain (GenBankAccession No. X66060).

FIG. 4 illustrates the mRNA sequence of Carica papaya caricain (GenBankAccession No. X69877).

DETAILED DESCRIPTION OF THE INVENTION Compositions

In one aspect of the present invention, there is provided a compositionfor the prophylaxis or treatment of a condition related to glutenintolerance, the composition including at least partially purifiedcaricain, or a biologically active fragment, analogue or variantthereof.

It has been found that caricain is capable of modifying toxicoligopeptides that are produced following gluten ingestion to producenon-toxic peptides, thereby offering an improved method of preventing orat least partly alleviating their toxic effect in gluten intolerantindividuals. Without being bound by theory, it is expected that theeffect is dose-dependent of the enzyme quantity and the amount ofingested gluten.

As used herein, the term “caricain” (EC 3.4.22.30) refers to a cysteineprotease typically found in the latex of plants such as Carica papaya.Other names by which caricain is known in the art include papapyaprotease omega, papaya endopeptidase III, papaya peptidase A, papayapeptidase II and papaya proteinase III. Caricain is a member of thepapain superfamily and is homologous to other plant and animal cysteineproteases. Caricain is naturally expressed as an inactive zymogen calledpro-caricain. The inactive form of the protease contains an inhibitorypro-region which consists of an additional 106 N-terminal amino acids.Studies have shown that the rate-limiting step in the in vitroactivation of procaricain is the dissociation of the prodomain, which isthen followed by proteolytic cleavage of the extended polypeptide chainof the proregion. The prodomain provides a stable scaffold which mayfacilitate the folding of the C-terminal lobe of procaricain (see Groveset al., 1996, Structure, 4(10):1193-1203). For the first time, thepresent inventors have identified caricain as a key enzyme in theconversion of toxic oligopeptides of gluten proteins such as wheat, tonon-toxic fragments.

In one embodiment, caricain is derived from Carica papaya and wasdiscovered by Schack in 1967 (Dubey K. at al. 2007, Papain-likeproteases: Applications of their inhibitors; African Journal ofBiotechnology 6 (9) 1077-1086).

In another embodiment, the caricain includes an amino acid sequence asshown in FIG. 1, or a biologically active fragment, analogue or variantthereof.

The composition according to the present invention may further includean intestinal enzyme extract, such as is described in internationalpatent application PCT/AU03/00633 (publication no. WO 2003/100051; thecontents of which are incorporated herein by reference). The presentinventors have identified, for the first time, synergism in thecombination of caricain and the intestinal enzyme extract described inWO 2003/100051.

At Least Partially Purified Caricain

In one embodiment of the present invention, caricain is at leastpartially purified from a natural source (e.g., papaya latex) inaccordance with conventional methods known to the skilled addressee. Ina certain embodiment of the present invention, caricain is at leastpartially purified from the latex of Carica papaya using any methodknown in the art, including, but not limited to, the methods describedin Azarkan M. et al. (“Fractionation and purification of the enzymesstored in the latex of Carica papaya”, J Chromatogr B Analyt TechnolBiomed Life Sci. 2003 790(1-2):229-38) and Buttle D. J. (Caricain InHandbook of Proteolytic Enzymes, 2 edition, p. 1130-1132, Elsevier,London).

Papaya, the fruit of the tree Carica papaya, in the genus Carica, isalso known as mama), tree melon, fruta bomba, lechosa or pawpaw. Methodsuseful for the isolation of caricain from a natural source such aspapaya latex include, but are not limited to, solid-liquid extraction,liquid-liquid extraction, solid-phase extraction, membrane filtration,ultrafiltration, dialysis, electrophoresis, solvent concentration,centrifugation, ultracentrifugation, liquid or gas phase chromatography(including size exclusion chromatography, affinity chromatography, etc)with or without high pressure, lyophilisation, evaporation,precipitation with various “carriers” (e.g., antibodies),crystallization, and any combination thereof. The skilled addresseewould understand how to use such options, in a sequential fashion, inorder to enrich each successive fraction for caricain by following itsactivity throughout the purification procedure. The activity of the atleast partially purified caricain can be measured using a variety ofmethods known to the skilled addressee, as herein described.

Solid-liquid extraction includes, but is not limited to, the use ofvarious solvents, vortex shakers, ultrasounds and other means to enhanceextraction, as well as recovery by filtration, centrifugation andrelated methods as described in the art (see, e.g., Cannell RJP, NaturalProducts Isolation, Humana Press, 1998). Examples of solvents that maybe used include, but are not limited to, hydrocarbon solvents,chlorinated solvents, organic esters, organic ethers, alcohols, water,and combinations thereof.

Liquid-liquid extraction includes, but is not limited to, the use ofsolvents known in the art such as hydrocarbon solvents, chlorinatedsolvents, organic esters, organic ethers, alcohols, water, variousaqueous solutions, and combinations thereof. The liquid-liquidextraction can be facilitated manually, or it can be automated(completely or in part), and the solvent can be removed and/orconcentrated by standard techniques in the art.

Membrane, reverse osmosis and ultrafiltration include, but are notlimited to, the use of various types of membranes known in the art, aswell as the use of pressure, vacuum, centrifugal force, and/or othermeans that can be utilised in membrane and ultrafiltration processes.

Dialysis typically includes the use of membranes having a molecularweight cut-off that is selective for the removal of various constituentsfrom the natural source so as to increase the relative purity ofcaricain in a sample. The present invention also encompassed therecovery of purified and/or fractionated extracts from either thedialysate or the retentate by various means known in the art including,but not limited to, lyophilization and crystallization.

Chromatography includes, but is not limited to, the use of regularcolumn chromatography, flash chromatography, high performance liquidchromatography (HPLC), medium pressure liquid chromatography (MPLC),supercritical fluid chromatography (SFC), countercurrent chromatography(CCC), moving bed chromatography, simulated moving bed chromatography,expanded bed chromatography, and planar chromatography. Examples ofsorbents that may be used in chromatography include, but are not limitedto, silica gel, alumina, fluorisil, cellulose and modified cellulose,various modified silica gels, ion-exchange resins, size exclusion gels,chemically modified gels, and other sorbents known to those skilled inthe art. The present invention also includes the use of two or more saltgradients to effect the fractionation and/or partial purification ofcaricain by chromatographic methods. When water or an aqueous phase isused, it may contain varying amounts of inorganic or organic salts,and/or the pH may be adjusted to different values with an acid or a basesuch that fractionation and/or purification is enhanced.

The process of at least partially purifying caricain from a naturalsource may also include the concentration of the purified or partiallypurified caricain by solvent removal of the original extract and/orfractionated extract, and/or purified extract. The techniques of solventremoval are known to those skilled in the art and include, but are notlimited to, rotary evaporation, distillation (normal and reducedpressure), centrifugal vacuum evaporation (speed-vac), lyophilizationand combinations thereof.

When referring to peptides, proteins and peptide analogs (e.g., caricainor bromelain) of the invention, the term “at least partially purified”typically means a composition which is partially to completely free ofother components (e.g., other proteins, nucleic acids, lipids,carbohydrates) with which the peptides, proteins or analogs areassociated in a non-purified, e.g., native state or environment. The atleast partially purified peptides and proteins can generally be in ahomogeneous or nearly homogenous state, although it can be either in adry state or in an aqueous solution. Purity and homogeneity aretypically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography. In one embodiment, peptides such as caricain can befurther purified using routine and well-known methods, such as thosedescribed herein.

In a certain embodiment of the present invention, the at least partiallypurified protein such as caricain can constitute at least about one or afew percent by weight of the total weight of the composition, forexample, at least about five percent by weight of the total weight ofthe composition. In another embodiment, the at least partially purifiedcaricain can constitute at least about ten percent by weight of thetotal weight of the composition. In another embodiment, the at leastpartially purified caricain constitutes at least about twenty percent byweight of the total weight of the composition. In another embodiment,the at least partially purified caricain can constitute at least aboutfifty percent by weight of the total weight of the composition. In afurther embodiment, the at least partially purified caricain canconstitute at least about eighty percent by weight of the total weightof the composition. In other embodiments, the at least partiallypurified caricain constitute at least about ninety percent or at leastabout ninety-five percent or more by weight of the total weight of thecomposition.

In other embodiments, the composition can be an oral composition thatcontains caricain at about 5% w/w to about 95% w/w, based on the totalweight of the oral composition.

Assay for Caricain Activity

Partially purified caricain can be tested for its ability to reduce thetoxicity of toxic gluten peptides by any one or more of the proceduresherein described. It will be understood by the skilled addressee thatmethods of isolating and purifying caricain from papaya latex, or fromany other suitable source, will be such that at least some enzymeactivity of the isolated peptidase is retained so as to provide for theprophylaxis or treatment of a condition arising from gluten intolerance.One skilled in the art would appreciate that there are numerous methodsand techniques for measuring qualitatively and/or quantitatively theability of the at least partially purified caricain to reduce thetoxicity of toxic gluten peptides, either in vitro or in vivo, as hereindescribed.

Caricain, as herein described, may be identified by its ability tomodify a substrate to inactivate toxic gluten oligopeptides, where thesubstrate may include, but is not limited to, a gliadin, hordein,secalin or avenin protein. Toxic gliadin oligopeptides include peptidesderived during normal human digestion of gliadins and related storageproteins, as herein described, from dietary cereals (e.g. wheat, rye,barley, and the like). Such oligopeptides are believed to act asantigens for T cells in patients suffering from a condition arising fromgluten intolerance, such as Celiac Sprue. For binding to Class II MHCproteins, immunogenic peptides are usually from about 8 to 20 aminoacids in length, more usually from about 10 to 18 amino acids. Suchpeptides may include QQPY or related tyrosine-containing motifs.Determination of whether an oligopeptide is immunogenic for a particularpatient is readily determined by standard T cell activation and otherassays known to those of skill in the art. The ability of caricain, or abiologically active fragment, analogue or variant thereof, as hereindescribed, to inactivate a toxic gluten peptide can be determined by anymethods known in the art, for example, by using a Rat Liver Lysosome(RLL) assay, as described, for example, in WO 2003/100051 and Cornelland Townley (1974; Gut, 15(11):862-869), both of which are incorporatedherein by reference.

In a certain embodiment, the caricain according to the present inventionis capable of modifying toxic peptides such as those containing PSQQ,QQQP and QQPY motifs, and the A-gliadin wheat peptides QNPSQQQPQ(residues 11-19), RPQQPYPQPQPQ (residues 75-86), LGQQQPFPPQQPY (residues31-43), PQPQPFPSQQPY (residues 44-55) and LGQGSFRPSQQN (residues206-217).

The ability of caricain to modify a substrate can be determined, forexample, by measuring the ability of an enzyme to increase theconcentration of free NH₂-termini in a reaction mixture containing 1mg/ml substrate and 10 mg/ml of caricain, incubated at 37° C. for 1hour. Caricain according to the present invention will increase theconcentration of the free amino termini under such conditions, usuallyby at least about 10%, more usually by at least about 25%, andpreferably by at least about 50%. Caricain according to the presentinvention may also be capable of reducing the toxicity of oligopeptidesgreater than about 1000 Da in a 0.1 ml of 50 mg/ml substrate after a 2hour incubation with 0.2 ml of 10 mg/ml of the peptidase by at leastabout 2-fold, usually by at least about 5-fold, and preferably by atleast about 10-fold. The toxicity of such oligopeptides can bedetermined by methods known in the art, for example, by using a RatLiver Lysosome (RLL) assay, as described, for example, in WO 2003/100051and Cornell and Townley (1973); Clin. Chim. Acta 49: 181-188 both ofwhich are incorporated herein by reference.

In one embodiment of the present invention, caricain will displaydetoxifying activity (as measured, e.g., by an RLL assay) of at leastabout 20%, optionally of at least about 50% or optionally of at leastabout 90% protection to the lysosomes.

In some embodiments of the present invention, the composition includesat least 15 mg of caricain, or a biologically active fragment, variantand analogue thereof.

Other Compositions

In another embodiment of the invention, compositions of interest mayinclude caricain in combination with other enzymes capable ofinactivating toxic gluten oligopeptides, including, but not limited tobromelain and an intestinal extract, such as is described ininternational patent application PCT/AU03/00633 (publication no. WO2003/100051, the contents of which are incorporated herein byreference).

It has been found by the present inventors that the combination ofcaricain with an intestinal enzyme extract (as described, e.g., in WO2003/100051) produces a surprisingly synergistic effect on the cleavageof toxic gliadin oligopeptides, as demonstrated, for example, in a RatLiver Lysosome Protection Assay.

Bromelain is a plant protease which is typically isolated from pineapple(Ananas comosus). It has an optimum pH range of 5 to 8 depending uponthe substrate with broad specificity for peptide bonds. Bromelain may bepurified from sources such as pineapple using any standard techniqueknown in the art, including, but not limited to, the method ofpurification described by Yamada F. et al. (“Purification andcharacterization of a proteinase from pineapple fruit, fruit bromelainFA2”; J Biochem (Tokyo). 1976; 79(6):1223-34). Isolated and purifiedbromelain is also available commercially.

Caricain has been found by the present inventors to elute with a highmolecular weight fraction of papaya latex (Fraction 4 in the 30-35 KDarange; see Examples section), consistent with the sequence of its activeform (Groves et al., 1996, Structure, vol 4:1193-1203). Furtherpurification of Fraction 4 by HPLC on Biosep SEC S 2000 confirmed thatthe early high molecular weight portion of Fraction 4 containedcaricain, glutamine cyclotransferase and chymopapain, but the latter twoenzymes do not appear to be significant contributors to detoxificationof gliadin. The applicant has shown that phenylmethyl sulfonyl fluoride(PMSF) does not significantly inhibit the action Fraction 4 on rat liverlysosomes at 6 mM concentration, confirming that the main enzymespresent in this fraction were not serine proteases.

The intestinal extract, as herein described, may be derived from anyportion of a gastrointestinal tract, including, but not limited to, theduodenum. The intestinal extract may be derived from any species, aslong as it displays an ability to detoxify toxic gliadin peptides alone,or in a synergistic capacity with a caricain. In a certain embodiment ofthe present invention, the intestinal extract in derived from porcineintestine.

Other animal forms of these proteins may be used, or modified forms maybe isolated from other commercially available sources.

The compositions of the present invention may also include otherenzymes, such as (but not limited to) fungal proteases from Aspergillusspp. such as Aspergillus oryzae (e.g. Byun et al. (2001) J. Agric. FoodChem. 49, 2061-2063) and Lactobacilli spp. such as Lactobacillushelveticus (e.g. Vesanto et al., (1995) Microbiol. 141, 3067-3075), andLactococcus lactis (Mayo et al., (1991) Appl. Environ. Microbiol. 57,38-44).

Biologically Active Fragments, Variants and Analogues Thereof

As used herein and with reference to caricain, the term “biologicallyactive fragment” typically refers to a fragment that retains its abilityto detoxify gluten peptides, in vitro or in vivo.

Peptidase fragments of interest include, but are not limited to,fragments of at least about 20 contiguous amino acids, more usually atleast about 50 contiguous amino acids, and may comprise 100 or moreamino acids, up to the complete protein, and may extend further toinclude additional sequences. In each case, the key criterion is whetherthe fragment retains the ability to modify the toxic oligopeptides thatcontribute to a condition arising from gluten intolerance.

As used herein, the term “native” preferably refers to caricain havingan amino acid sequence that occurs in nature (e.g., a natural protein).Such fragments may generally be identified using techniques well knownto those skilled in the art in identifying peptidase activity, forexample, as hereinbefore described.

As used herein and with reference to bromelain, the term “biologicallyactive fragment” typically refers to a fragment of bromelain thatretains its ability to contribute to the detoxification of glutenpeptides in combination with caricain, or a biologically activefragment, analogue or variant thereof.

As used herein and with reference to caricain, the term “analogue”typically denotes a peptidase that has an amino acid sequence that issubstantially identical to the amino acid sequence of the naturallyoccurring caricain. As used herein and with reference to bromelain, theterm “analogue” typically denotes a peptidase that has an amino acidsequence that is substantially identical to the amino acid sequence ofthe respective naturally occurring enzyme.

The term “substantially identical”, as used in regards to an analogue,typically denotes a substitution or addition of one or more amino acidssuch that the resulting analogue has at least some of the biologicalactivity of the naturally occurring enzyme. Analogues may be naturallyoccurring, such as an allelic variant or an mRNA splice variant, or theymay be constructed using synthetic or recombinant techniques availableto one skilled in the art.

As used herein and with reference to caricain and bromelain, the term“variant” typically denotes an enzyme that exhibits an amino acidsequence that is at least 80% identical to the native enzyme. Alsocontemplated are embodiments in which a variant comprises an amino acidsequence that is at least 90% identical, optionally at least 95%identical, optionally at least 98% identical, optionally at least 99%identical, or optionally at least 99.9% identical to the nativemolecule. Percent identity may be determined by visual inspection and/ormathematical calculation by methods known to those skilled in the art.Variants may be naturally occurring, synthetic or recombinant.

In one embodiment of the present invention, a variant of caricain orbromelain includes an enzyme that is substantially homologous to thenative form of the enzyme, but which has an amino acid sequencedifferent from that of the native form because of one or more deletions,insertions or substitutions. Certain embodiments include amino acidsthat comprise from one to ten deletions, insertions or substitutions ofamino acid residues when compared to a native sequence. A given sequencemay be replaced, for example, by a residue having similar physiochemicalcharacteristics. Examples of such conservative substitution of onealiphatic residue for another, such as Ile, Val, Leu or Ala for oneanother; substitution of one polar residue for another, such as betweenLys and Arg, or Glu and Asp, or Gln and Asn; or substitutions of onearomatic residue for another, such as Phe, Trp or Tyr for one another.Other conservative substitutions, e.g., involving substitutions ofentire regions having similar hydrophobicity characteristics, are wellknown in the art. Variants may also be generated by the truncation of anative peptidase amino acid. Further variants encompassed by the presentinvention include, but are not limited to, deglycosylated amino acids,or fragments thereof, or those amino acids demonstrating increasedglycosylation when compared to the native enzyme.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan). Thus, an amino acid residueof caricain is preferably replaced with another amino acid residue fromthe same side chain family. In a preferred embodiment, mutations can beintroduced randomly along all or part of the enzyme coding sequence,such as by saturation mutagenesis. The resultant mutants can be screenedto identify variants that demonstrate at least some of the biologicalactivity of the native enzyme. Following mutagenesis, the encodedprotein can be expressed recombinantly and the activity of the enzymecan be determined by the methods described herein.

Also envisaged are modifications that do not alter the primary sequenceof the native form of caricain or bromelain, including, but not limitedto, chemical derivatization of proteins (e.g., acetylation orcarboxylation), glycosylation (e.g., those made by modifying theglycosylation patterns of a protein during its synthesis and processingor in further processing steps), as well as sequences that havephosphorylated amino acid residues (e.g., phosphotyrosine,phosphoserine, or phosphothreonine).

Also useful in the practice of the present invention is caricain orbromelain that has been modified using molecular biological techniquesand/or chemistry so as to improve their resistance to proteolyticdegradation and/or to acidic conditions such as those found in thestomach, and to optimize solubility properties or to render them moresuitable as a therapeutic agent. Analogs of such proteins include thosecontaining residues other than naturally occurring L-amino acids (e.g.,D-amino acids or non-naturally occurring synthetic amino acids).

The caricain according to the present invention may be prepared by invitro synthesis using conventional methods as known in the art. Variouscommercial synthetic apparatuses are available, for example, automatedsynthesizers (e.g., CS936X Peptide Synthesizer, CSBio Company, Inc.).Using such synthesizers, a skilled person can readily substitute for thenaturally occurring amino acids one or more unnatural amino acids. Theparticular sequence and the manner of preparation will be determined byconvenience, economics, purity required, and the like. If desired,various groups can be introduced into the protein during synthesis thatallow for linking to other molecules or to a surface. For example,cysteines can be used to make thioethers, histidines can be used forlinking to a metal ion complex, carboxyl groups can be used for formingamides or esters, amino groups can be used for forming amides, and thelike.

Nucleic Acid Molecules

In yet another aspect of the present invention, there is provided acomposition for the prophylaxis or treatment of a condition arising fromgluten intolerance, the composition including a nucleic acid moleculehaving a nucleic acid sequence that encodes caricain, or a biologicallyactive fragment, analogue or variant thereof. The nucleic acid moleculeshould be capable of driving the expression of a recombinant analogue ofcaricain, including a biologically active fragment, analogue or variantthereof. For example, and without being bound by theory, the nucleicacid molecule may transfect a cell lining the gastrointestinal tract ofa subject into whom it has been administered, where it becomesincorporated into the subject's genome. The incorporation of the nucleicacid molecule will then result in the expression of the recombinantcaricain, or a biologically active fragment, analogue or variantthereof.

As used herein, the term “nucleic acid molecule” includes DNA molecules(e.g., a cDNA or genomic DNA) and RNA molecules (e.g., an mRNA) andanalogs of the DNA or RNA generated, for example, by the use ofnucleotide analogues. The nucleic acid molecule can be single-strandedor double-stranded, but preferably is double-stranded DNA. Encompassedby the present invention are naturally occurring and synthetic nucleicacid molecules, or combinations thereof, whose nucleic acid sequenceencodes caricain, or a biologically active fragment, analogue or variantthereof, as hereinbefore described.

As used herein, a “naturally-occurring” nucleic acid molecule typicallyrefers to an RNA or DNA molecule having a nucleotide sequence thatoccurs in nature (e.g., as found in a papaya plant).

As used herein, the terms “gene” and “recombinant gene” preferably referto nucleic acid molecules which include an open reading frame encoding apeptidase as herein described, and can further include non-codingregulatory sequences, and introns.

For example, the nucleic acid molecule encoding caricain includes anucleotide sequence which is about 65% to about 99% or more homologousto a nucleotide sequence encoding a naturally-occurring caricain. Thenucleic acid molecule encoding caricain may be derived from any source,including, but not limited to papaya, whereas the analogues envisaged asbeing within the spirit of the present invention may be derived fromnon-plant species, including, but not limited to, human, porcine. ovineand bovine.

In a certain embodiment of the present invention, the nucleic acidmolecule includes a nucleic acid sequence as shown in FIG. 2, or afunctional equivalent thereof.

As used herein, the term “functional equivalent thereof” refers to asequence that has an analogous function to the sequence of which it is afunctional equivalent. By “analogous function” is meant that thesequences share a common function, for example, in encoding caricain, ora biologically active fragment, analogue or variant thereof. In someembodiments, a functionally equivalent sequence may exhibit sequenceidentity with the sequence of which it is a functional equivalent. Thesequence identity between the functional equivalent and the sequence ofwhich it is a functional equivalent may be at least 50% across thelength of the functional equivalent, at least 60% across the length ofthe functional equivalent or greater than 70%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% across the length of the functional equivalent.

In another embodiment of the present invention, the nucleic acidmolecule encodes an amino acid sequence as shown in FIG. 1, or abiologically active fragment, analogue or variant thereof, as hereindescribed. The nucleic acid molecules of the invention, as hereindescribed, can be inserted into vectors and used as gene therapyvectors.

In yet another embodiment of the present invention, there is provided acomposition, as herein described, further including a nucleic acidmolecule that encodes recombinant bromelain, or a biologically activefragment, analogue or variant thereof, as herein described.

Host Cells

In yet another aspect of the present invention, there is provided acomposition for the prophylaxis or treatment of a condition related togluten intolerance, the composition including a host cell, wherein thehost cell is capable of expressing recombinant caricain, or abiologically active fragment, analogue or variant thereof, as hereindescribed. In certain embodiments of the present invention, the hostcell is a eukaryotic cell or cell line of any species selected from thegroup including embryonic stem cells, embryonic carcinoma cells,hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, bronchial epithelial cells and immunecells. The host cell may also be of a lower organism such as bacteria.In one embodiment, the host cell may be a microorganism that cancolonise the gastrointestinal tract of the recipient, such as, but notlimited to, Lactobacillus spp.

In another embodiment of the present invention, the host cell is a plantengineered to express recombinant caricain and/or bromelain, orbiologically active fragments, analogues or variants thereof, as hereindescribed. For example, the host cell may be an edible plant, wherein asubject in need thereof (e.g., a subject showing, or at risk of showing,symptoms of gluten intolerance) is able to ingest the edible plant,thereby administering a dose of the recombinant caricain and/orbromelain (or biologically active fragments, analogues or variantsthereof, as herein described). As used herein, the term “plant” includesreference to whole plants, plant parts or organs (e.g., leaves, stems,roots, etc.), plant cells, seeds and progeny of same. The term “plantcell”, as used herein, further includes, without limitation, cellsobtained from or found in: seeds, suspension cultures, embryos,meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen, and microspores. Plant cells can alsobe understood to include engineered cells, such as protoplasts, obtainedfrom the aforementioned tissues. The class of plants which can be usedin accordance with the present invention is generally as broad as theclass of higher plants amenable to transformation techniques, includingboth monocotyledonous and dicotyledonous plants. Particularly preferredplants include maize, soybean, sunflower, sorghum, canola, alfalfa,cotton, rice, barley, and millet.

Any suitable method may be used to engineer a plant cell to expressrecombinant caricain and/or bromelain (or biologically active fragments,analogues or variants thereof, as herein described), including, but notlimited to, the methods described in U.S. Pat. No. 6,977,325 (thecontents of which are incorporated herein be reference).

The composition may further include a host cell that is capable ofexpressing recombinant bromelain, or a biologically active fragment,analogue or variant thereof, as herein described. For example, in acertain embodiment of the present invention, the composition includes ahost cell that expresses recombinant caricain (or a biologically activefragment, analogue or variant thereof, as herein described) andrecombinant bromelain (or a biologically active fragment, analogue orvariant thereof, as herein described). In another embodiment, thecomposition of the present invention includes a first host cell thatexpresses recombinant caricain (or a biologically active fragment,analogue or variant thereof, as herein described) and a second host cellthat expresses recombinant bromelain (or a biologically active fragment,analogue or variant thereof, as herein described).

Pharmaceutical Compositions

The compositions according to the present invention, as hereinbeforedescribed, may be in the form of a pharmaceutical composition, in whichthe composition further includes a pharmaceutically acceptable carrier,excipient, diluent and/or adjuvant.

Pharmaceutical compositions of the present invention may be employedalone or in conjunction with other compounds, such as therapeuticcompounds.

As used herein, the phrase “pharmaceutically acceptable carrier”includes, but is not limited to, solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Typically, the route of administrationis parenteral, including oral (e.g., ingestion, inhalation) or rectal.Solutions or suspensions used for parenteral application can include oneor more of the following components: a sterile diluent such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents; antibacterial agents suchas methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide.

Generally, the pharmaceutical composition is stable under the conditionsof manufacture and storage and preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, or liquid polyetheyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof a dispersion or by the use of surfactants. Prevention of the actionof microorganisms can be achieved by incorporation of variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugar, sodium chloride or polyalcohols such as mannitol, or sorbitol, inthe composition.

Oral compositions generally comprise an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or modified corn starch; a lubricant such as magnesiumstearate or other stearates; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or aflavouring agent such as peppermint, methyl salicylate, or orangeflavouring.

In one embodiment, the compositions of the present invention areprepared with carriers that will protect the compositions according tothe present invention against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art.Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.“Dosage unit form”, as used herein, refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The toxicity and therapeutic efficacy of suchcompounds can be determined by standard pharmaceutical proceduresincluding in vitro assays, cell cultures or experimental animals, e.g.,for determining the LD₅₀ (the dose lethal to 50% of the population) andthe ED₅₀ (the dose therapeutically effective in 50% of the population)depending on the compound studied. The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD₅₀/ED₅₀. Compounds which exhibit high therapeutic indicesare preferred. While compounds that exhibit toxic side effects may beused, care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the in vitro studies, cell culture assays andanimal studies can be used in formulating a range of dosages for use inhumans. The dosage lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. The therapeuticallyeffective dose of an enzyme can be estimated initially from in vitroassays. Such information can be used to more accurately determine usefuldoses in humans. In one embodiment of the present invention, theeffective dose of caricain is at least about 0.1 mg per kg body weighttaken with each meal for adults and typically half that dose forchildren.

Depending on the patient and condition being treated and on theadministration route, caricain, or a biologically active fragment,variant and analogue thereof, may be administered in dosages of about0.1 mg to about 500 mg/kg body weight per day, e.g. about 15 mg/day foran average person. For instance, a typical dose of caricain, or abiologically active fragment, analogue or variant thereof, in patientswill be in at least about 1 mg/adult, more usually at least about 15 mg;and preferably at least about 50 mg; and preferably not more than about500 mg. Dosages will be appropriately adjusted for pediatricformulation. In children the effective dose may be lower, for example atleast about 0.1 mg, or 0.5 mg of caricain, or a biologically activefragment, variant and analogue thereof. In combination therapyinvolving, for example, caricain and intestinal prolidase, a comparabledose of the composition may be given; however, the ratio will beinfluenced by the relative stability of the composition toward gastricinactivation. The caricain may be present in the composition of thepresent invention as an at least partially purified extract, or as asynthesized pharmaceutically acceptable protein.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific enzyme, the severity of the symptomsand the susceptibility of the subject to side effects. In someembodiments, dosages for a given enzyme are readily determinable bythose of skill in the art by a variety of means. An exemplary means isto measure the biological activity of a given compound required toovercome the symptoms.

In some embodiments of the present invention, the composition of thepresent invention includes at least 15 mg caricain, or a biologicallyactive fragment, variant and analogue thereof.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingthe activity of the specific compound employed, the age, body weight,general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, the degree of expression or activity to be modulated,sensitivity to gluten, previous treatments and other diseases present.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

For oral preparations, the compositions according to the presentinvention can be used alone or in combination with appropriate additivesto make tablets, powders, granules or capsules, for example, withconventional additives, such as lactose, mannitol, corn starch or potatostarch; with binders, such as crystalline cellulose, cellulose variants,acacia, corn starch or gelatins; with disintegrators, such as cornstarch, potato starch or sodium carboxymethylcellulose; with lubricants,such as talc or magnesium stearate; and if desired, with diluents,buffering agents, moistening agents, preservatives and flavoring agents.

In one embodiment of the present invention, the oral formulationscomprise enteric coatings, so that the active agent is delivered to theintestinal tract. Enteric formulations are often used to protect anactive ingredient from the strongly acid contents of the stomach. Suchformulations can be created by coating a solid dosage form with a filmof a polymer that is insoluble in acid environments, and soluble inbasic environments. Exemplary films are cellulose acetate phthalate,polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate succinate, methacrylatecopolymers, cellulose acetate phthalate and acrylic coating systems suchas Acryl-Ease (Colorcon).

Other enteric formulations comprise engineered polymer microspheres madeof biologically erodable polymers, which display strong adhesiveinteractions with gastrointestinal mucus and cellular linings and cantraverse both the mucosal absorptive epithelium and thefollicle-associated epithelium covering the lymphoid tissue of Peyer'spatches. The polymers maintain contact with intestinal epithelium forextended periods of time and actually penetrate it, through and betweencells (see, for example, Mathiowitz et al. (1997) Nature 386 (6623):410-414. Drug delivery systems can also utilize a core of superporoushydrogels (SPH) and SPH composite (SPHC), as described by Dorkoosh etal. (2001) J Control Release 71 (3):307-18).

Methods of Prophylaxis or Treatment

In another aspect of the present invention, there is provided a methodfor the prophylaxis or treatment of a condition related to glutenintolerance, the method including administering to a subject in needthereof a composition according to the present invention, as hereindescribed.

In some embodiments of the present invention, the method includesadministering to the subject in need thereof at least 15 mg of caricain,or a biologically active fragment, variant and analogue thereof. In someembodiments of the present invention, the method includes administeringto the subject in need thereof at least 15 mg of caricain, or abiologically active fragment, variant and analogue thereof before ameal, following a meal or with a meal.

The method of the present invention can be used for prophylaxis orsafeguarding, as well as for therapeutic purposes. Accordingly, as usedherein, the term “treatment” and the like refers to any diminution inthe severity of a pre-existing disease, condition or symptom of glutenintolerance, particularly as measured by the severity of symptoms suchas, but not limited to, fatigue, chronic diarrhoea, and malabsorption ofnutrients, weight loss, abdominal distension and anaemia. As usedherein, the term “prophylaxis” and the like refer to the prevention of adisease, condition or symptom of gluten intolerance. Other indices ofgluten intolerance include, but are not limited to, the presence ofantibodies specific for glutens, the presence of antibodies specific fortissue transglutaminase, the presence of pro-inflammatory T cells andcytokines, damage to the villous structure of the small intestine asevidenced by histological or other examination, enhanced intestinalpermeability, and the like.

Subjects that can benefit from the methods of the present invention maybe of any age and include adults and children. Children in particularbenefit from prophylactic treatment, as prevention of early exposure totoxic gluten peptides can prevent initial development of the disease.Children suitable for prophylaxis can be identified by genetic testingfor predisposition, for example, by HLA typing, by family history, by Tcell assay, or by other means known to the skilled addressee.

In one embodiment of the present invention, the method of prophylaxis ortreatment further includes administering to the subject in need thereofan effective dose of an at least partially purified caricain, or abiologically active fragment, analogue or variant thereof, as hereindescribed.

In another embodiment of the present invention, the method ofprophylaxis or treatment further includes administering to the subjectin need thereof an effective dose of bromelain (or a biologically activefragment, analogue or variant thereof, as herein described), anintestinal extract (as hereinbefore described), or any combinationthereof. Caricain may be administered together with bromelain and/or anintestinal extract or as separate dosages, as required.

In a further aspect of the present invention, the method of prophylaxisor treatment includes administering to a subject in need thereof anucleic acid molecule including a nucleic acid sequence that encodesrecombinant caricain or a biologically active fragment, analogue orvariant thereof, as herein described. The methods of the presentinvention may further include administering to the subject a nucleicacid molecule including a nucleic acid sequence that encodes recombinantbromelain (or a biologically active fragment, analogue or variantthereof, as herein described).

In yet another aspect of the present invention, the method ofprophylaxis or treatment includes administering to a subject in needthereof a host cell that is capable of expressing in the subject arecombinant caricain, or an analogue or variant thereof, as hereindescribed. The methods of the present invention may further includeadministering to the subject a host cell that is capable of expressingrecombinant bromelain (or a biologically active fragment, analogue orvariant thereof, as herein described).

The methods according to the present invention may also be performed incombination with other modalities, including, but not limited to,administering to a subject in need thereof, an inhibitor of tissuetransglutaminase, an anti-inflammatory agent, an anti-ulcer agent, amast cell-stabilizing agents, and/or and an-allergy agent. Examples ofsuch agents include HMG-CoA reductase inhibitors with anti-inflammatoryproperties such as compactin, lovastatin, simvastatin, pravastatin andatorvastatin; anti-allergic histamine H1 receptor antagonists such asacrivastine, cetirizine, desloratadine, ebastine, fexofenadine,levocetirizine, loratadine and mizolastine; leukotriene receptorantagonists such as montelukast and zafirlukast; COX2 inhibitors such ascelecoxib and rofecoxib; p38 MAP kinase inhibitors such as BIRB-796; andmast cell stabilizing agents such as sodium chromoglycate (chromolyn),pemirolast, proxicromil, repirinast, doxantrazole, amlexanox nedocromiland probicromil.

Various methods for administration may be employed, preferably usingoral administration, for example with meals. The dosage of thetherapeutic formulation will vary widely, depending upon the nature ofthe disease, the frequency of administration, the manner ofadministration, the clearance of the agent from the host, and the like.The initial dose can be larger, followed by smaller maintenance doses.The dose can be administered as infrequently as weekly or biweekly, ormore often fractionated into smaller doses and administered daily, withmeals, semi-weekly, or otherwise as needed to maintain an effectivedosage level.

The therapeutic effect can be measured in terms of clinical outcome orcan be determined by immunological or biochemical tests. Suppression ofthe deleterious T-cell activity can be measured by enumeration ofreactive Th1 cells, by measuring the release of cytokines at the sitesof lesions, or using other assays for the presence of autoimmune T cellsknown in the art. Alternatively, one can look for a reduction inseverity of the symptoms of the disease.

Preparation of Food Articles

In another aspect of the present invention, there is provided a methodof preparing an article of food derived from a gluten-containingmaterial, the method including treating the article of food or thegluten-containing material with caricain, or a biologically activefragment, analogue or variant thereof, so as to reduce the amount oftoxic gluten-derived oligopeptides present in the article of food.

In yet another aspect of the present invention, the method furtherincludes treating the article of food or the gluten-containing materialwith bromelain (or a biologically active fragment, analogue or variantthereof, as herein described).

The discussion of documents, acts, materials, devices, articles and thelike is included in this specification solely for the purpose ofproviding a context for the present invention. It is not suggested orrepresented that any or all of these matters formed part of the priorart base or were common general knowledge in the field relevant to thepresent invention before the priority date of each claim of thisapplication.

Finally it is to be understood that various other modifications and/oralterations may be made without departing from the spirit of the presentinvention as outlined herein.

Compositions and their use in the prophylaxis or treatment of conditionsarising from gluten intolerance according to certain embodiments of thepresent invention will now be described in the following examples. Itshould be understood, however, that the following description isillustrative only and should not be taken in any way as a restriction onthe generality of the invention described above.

EXAMPLES

Materials

Crude caricain was extracted (see Example 2) from dry Papaya latex(Enzyme Solutions). Papaya latex is a food grade mix of proteolyticenzymes isolated from papaya fruit. In two Rat Liver Lysosome (RLL)assays, crude caricain offered good protection of approximately 92%-94%at a concentration of about 10 mg/ml, using the normal amount of gliadindigest which is 5 mg for the assay.

The Rat Liver Lysosome (RLL) assay, as described, for example, inCornell and Townley (1973; Clinica Chimica Acta 49:181-188) and Cornelland Townley (1974; Gut, 15(11):862-869), is based on the fact that apeptic-tryptic-pancreatinic digest of wheat gliadin disrupts rat liverlysosomes, causing a reduction in optical density (at 400 nm) of asuspension of these organelles. This is evidence of a cytotoxic reactionby the peptides present in the digest. However, if the enzyme extract ispre-incubated with the toxic gliadin digest for two hours, the change inoptical density is much less after incubation with the lysosomes. Bycomparing a control (no toxic digest), a toxic sample (lysosomesincubated with a toxic digest of gliadin) and an extract treated sample(toxic digest of gliadin pre-incubated with enzyme extract prior toaddition of lysosomes), the extent of protection can be determined. Aprotection index (P.I.) can be calculated from:

${P.I.\; (\%)} = {\frac{{\% \mspace{20mu} {reduction}\mspace{14mu} {toxic}\mspace{14mu} {sample}} - {\% \mspace{14mu} {reduction}\mspace{14mu} {extract}\mspace{14mu} {treated}\mspace{14mu} {sample}}}{\% \mspace{14mu} {reduction}\mspace{14mu} {toxic}\mspace{14mu} {sample}} \times 100}$

Example 1: Fractionation Experiments

To characterise caricain further, papaya latex was fractionated on anion-exchange Carboxymethyl (CM) Sephadex C-50 column and on a sizeexclusion Sephacryl S-300 column.

The ion-exchange column (3.2×20 cm) was equilibrated with 0.02Mphosphate buffer pH4.6 and 2 g of the crude papain applied in thestarting buffer (30 ml). After the unabsorbed fraction was obtained,other fractions were eluted by increasing the pH by application of a0.05 M phosphate buffer of pH6.8 and then by application of saltcontaining buffers of the same pH but containing 0.1M, 0.3M and 0.7Msodium chloride. The fractions were dialysed against distilled water andfreeze-dried. They were then assayed using the RLL assay at aconcentration of 10 mg/ml. The results are shown in Table 1 below:

TABLE 1 Ion exchange chromatography Fraction Yield % Elution conditionsProtection Index (%) 1 9.4 Unabsorbed 40 2 2.0 change of pH 53 3 20.00.1-0.3M NaCl 70 4 15.0 0.7M NaCl 84

The fractions collected from both the ion-exchange chromatography andthe size exclusion chromatography were subsequently tested by both anRLL assay (as herein described, for example, by reference to WO2003/100051 and Cornell and Townley (1973; Clinica Chimica Acta49:181-188) the contents of which are incorporated herein by reference)and a PEP assay (as described, for example, Marti et al. Prolylendopeptidase-mediated destruction of T cell epitopes in whole gluten:chemical and immunological characterization. J Pharmacol Exp Ther. 2005,312(1):19-26; the contents of which are incorporated herein byreference) to characterise the active fractions of papaya latex. The PEPassay uses Z-glycylproline-4-nitro-anilide as substrate to measure therate of attack on the C-terminal side of the proline residue.

The size exclusion Sephacryl S-300 column (88×2.2 cm) was equilibratedin pH 5.2 phosphate buffered saline and a sample of 1.2 g papaya latexin the buffer (10 ml) was applied carefully before continuing elution atthe rate of 30 ml/hour. Fractions of 15 ml were collected, adjusted topH 7.5 with 1M sodium hydroxide and assayed directly using 0.2 mlaliquots of each fraction. The results obtained are shown in Table 2.

TABLE 2 Sephacryl S300 fractions tested by RLL assay: Sample ProtectionIndex (%) Fraction 1 8 Fraction 2 1 Fraction 3 8 Fraction 4 58 Fraction5 64 Fraction 6 99 Fraction 7 100 Fraction 8 75 Fraction 9 29 Fraction10 19

As illustrated in Table 2, above, fractions 6 and 7 offered the highestprotection value and corresponded to proteins of approximately 30,000Daltons MW. Fraction 5 contained the largest amount of prolylendopeptidase (PEP) while Fraction 8 had the largest amount ofproteases, as measured by the Benzoyl-arginine ethyl ester (BAEE) assay(see, for example, Arnon R. (1970) Methods in Enzymology, XIX, 226-228;the contents of which are incorporated herein by reference). Theselatter enzymes are normally reported as having molecular weight of about23,000 Daltons.

Example 2: Synergistic Effect of Plant/Animal Derived Enzymes

A pig intestinal enzyme extract (as described, for example, in WO2003/100051) was combined with a crude caricain preparation to assesswhether there is a synergistic effect on their ability to detoxify toxicgluten peptides, as determined using the RRL assay, as hereinbeforedescribed. Crude caricain was prepared by dissolving papaya latexextract in water, adjusting the concentration of ammonium sulphate to60%, collecting the resulting precipitate by filtration, dialysing itand freeze-drying. This material was further enriched by chromatographyon CM Sephadex using phosphate buffers with elution of the crude QC with0.7 M sodium chloride, followed by dialysis and freeze-drying. Theenzyme preparations were assayed using the RLL assay at a concentrationof 6 mg/ml.

Pig intestinal Crude Combination extract Caricain (1:1) (6 mg/ml) (6mg/ml) (6 mg/ml) Protection Index (%) 61 71 92* *This is about the sameprotection offered by 10 mg/ml of crude papain alone.

Example 3: Heat and Acid Resistance

Caricain has been shown to retain around 70% activity of its activityafter exposure to heat or an acidic environment. For instance, theapplicant has shown that caricain is highly resistant to heat throughtheir observation that Fraction 4 from CM Sephadex chromatographyretained about 80% of its activity after heating to 85° C. Furthermore,when Fraction 4 was treated with 0.05M hydrochloric acid for 15 hours,it retained about 70% of its activity, demonstrating that caricain isalso acid resistant.

Example 4: Proteomics Analysis

The applicant has shown through mass spectrometry analysis that caricainas a major protein in Fraction 4 referred to in Example 1 (Table 1)above.

Number of peptides Protein identified identified Caricain precursor (EC3.4.22.30) 17 (38% coverage) Chymopapain precursor (EC 3.4.22.6) 6 (18%coverage) Glutamine cyclotransferase precursor (EC 2.3.2.5) 2 (7%coverage)

The proteomics analysis was performed by the Joint Proteomics ServicesFacility at the Ludwig Institute for Cancer Research and The Walter andEliza Hall Institute for Medical Research (Melbourne, Australia) usingthe following parameters:

For publication of these results, the following information is requiredby journals: Information on MS/MS database search Peaklist generatingsoftware extract_msn (Version 2.0, Thermoa Fisher Scientific) Parametersused Minimum mass m/z 700, Maximum mass m/z 5000. Grouping tolerance,1.5. Intermediate scans, 1. Minimum scans per group, 1. Precursorcharge, AUTO Minimum peaks in .DTA, 10. Search engine Mascot algorithm(Version 2.1.04, Matrix Science) Search Parameters Enzyme specificityconsidered Semi-trypsin. Considers all tryptic peptides as well as thosethat show tryptic specificity (KR) at one terminus, but where the otherterminus may be a non-tryptic cleavage. # of missed cleavages permitted3. Considers partial fragments where the digest was not complete. FixedModifications +58 Da for Carboxymethyl (Cysteine) if iodoacetic acid wasused for the alkylation process. +57 Da for Carbamidomethyl (Cysteine)if iodacetamide was used for the alkylation process. VariableModifications +42 Da for Acetylation (N-terminus) and +16 Da forOxidation (Methionine). Mass tolerance for precursor ions For LC-ESI IonTrap MS data, +/−2.0 Da. Mass tolerance for fragment ions For LC-ESI IonTrap MS/MS data, +/−0.5 Da. Database searched LudwigNR (Version Q12007). The latest protein nonredundant database produced by the Officeof Information Technology of the Ludwig Institute for Cancer Researchand obtained from the Swiss Institute of Bioinformatics (Moritz et al,Anal. Chem. 2004 76: 4811-24). Species restriction None. # proteinentries actually searched All LudwigNR proteins (i.e., currently over 4million proteins).

Example 5: Proteolytic Activity of Purified Caricain

A sample of purified caricain was supplied by The Free University ofBrussels, Belgium (courtesy of Professor Yvan Looze; see Azarkan M etal., “Fractionation and purification of the enzymes stored in the latexof Carica papaya”. J Chromatogr B Analyt Technol Biomed Life Sci (2003)790:229-238). The applicant has shown that this sample of purifiedcaricain was highly active in the Rat Liver Lysosome (RLL) Assay, asherein described, as compared to crude caricain.

Calculations:

Protection offered against gliadin in RLL Assay=32.3%Specific activity=32.3/0.0043=7512 (where 0.0043 is the number ofmilligrams in the assay; i.e., 4.3 micrograms).The specific activity of crude caricain was 40. Therefore, thefold-purification achieved was =7512/40=188 (being the ratio of specificactivities).

Thus, the activity of the purified caricain was approximately 190 timesthat of crude caricain and was active even at levels as low as 4micrograms in the RLL Assay. The results show that caricain contributesmost to the detoxification of gliadin in these studies.

Example 6: Use of Caricain Extract in the Treatment of SymptomsAssociated with Gluten Intolerance and Coeliac Disease

A male of 45 years of age diagnosed with coeliac disease by biopsy wastreated for his symptoms by taking tablets of caricain extract for aperiod of six weeks during which time he took one tablet (15 mg ofcaricain per tablet) at the start of each meal. The patient kept a dailyrecord of all symptoms and graded them according to severity (0=nosymptoms; 3=mild symptoms; 6=moderate symptoms). These results werecompared against those from a six week period before the trial periodduring which time the patient recorded his symptoms in the same way.

The results showed a reduction in severity from 307 symptom points priorto the commencement of treatment to 86 symptom points (72% reduction) atthe conclusion of treatment. Apart from the large reduction in symptomseverity, the patient also experienced symptom-free days and respondedthat the tablets took the worry out of dining out. Exposure to glutenchanged former episodes of diarrhoea to episodes of slight nausea whenthe tablets were used.

Example 7: Use of Caricain Extract in the Treatment of SymptomsAssociated with Gluten Intolerance and Dermatitis Herpatiformis (DH)

A 48 year old male patient diagnosed with DH by biopsy was exposed for 7days to a diet comprising breakfast cereal and toast and a lunchinclusive of a sandwich until he developed a moderately painful itch.After allowing three weeks for the itch to subside, the patient wasexposed to the same diet for 7 days during which time he was alsotreated with a caricain extract in tablet form (15 mg of caricain pertablet), taken with each meal of breakfast and lunch. The patient notedthat only a few mild itches developed while he was taking the caricainextract tablets and commented that they were most useful in alleviatingthe symptoms of his disease. From experience, he had noted that anyfurther gluten challenge without the tablets would soon have produced apainful rash, whereas, on the caricain extract tablets, the mild andless frequent itch was considered less likely to develop to the stage ofbecoming a painful rash. The results demonstrated that sufferers of DHare likely to find that treatment with caricain can provide a safeguardagainst accidental ingestion of gluten or to help them tolerate smallamounts of gluten without developing severe symptoms.

Future patent applications may be filed on the basis of or claimingpriority from the present application. It is to be understood that thefollowing claims are not intended to limit the scope of what may beclaimed in any such future applications. Features may be added to oromitted from the claims at a later date so as to further define orre-define the invention or inventions.

1. A method for the prophylaxis or treatment of gluten intolerance, themethod comprising administering to a subject in need thereof an oralcomposition that contains a first component comprising caricain at about5% w/w to about 95% w/w, based on the total weight of the firstcomponent of the oral composition, and a second component comprising atleast one pharmaceutically acceptable carrier, excipient, or diluent. 2.The method according to claim 1, wherein the first or second componentcomprises one or more additional enzymes capable of inactivating toxicgluten oligopeptides.
 3. The method of claim 2, wherein the one or moreadditional enzymes capable of inactivating toxic gluten oligopeptides isselected from bromelain or an intestinal extract.
 4. The methodaccording to claim 1, wherein the caricain is derived from Caricapapaya.
 5. The method according to claim 1, wherein the caricain has anamino acid sequence as shown in FIG. 1 or
 2. 6. The method according toclaim 1, wherein the composition is in the form of one or moreenterically coated tablets or capsules.
 7. The method according to claim6, wherein the one or more enterically coated tablets or capsulescontain at least 15 mg of caricain.